1. Field of the Invention
The present invention relates to methods and compositions useful in treating disorders in which the direct cause of the clinical disorder is the expression in the primary diseased cells of a differentiation program that does not normally exist. Such disorders are hereinafter referred to as Aberrant Programming (AP) Diseases. The invention also relates to method and compositions useful in therapeutically reprogramming normal cells.
As will be discussed more fully hereinafter, the AP diseases of this invention constitute a new disease classification and there is presented a novel molecular model of pathogenesis for these diseases. According to the molecular model of this invention, the basic disease causing entity in the AP diseases is a specific type of relational alteration among certain cellular components involved in program control. It is unlike any previously described molecular pathogenic mechanism. This model defines the nature of the therapy for these diseases, limits the potential set of therapeutically useful targets to a relatively small number of genes and leads to the unobvious conclusion that this includes the manipulation of certain “normal” genes is an appropriate approach for the treatment of AP diseases, thus, leading to a unique approach to therapy for the AP diseases of this invention. This model makes the selection of targets for proposed therapy straightforward and accessible to anyone skilled in the art. Also provided herein is a novel approach to diagnosing and developing prognostic criteria for the Aberrant Programming Diseases.
A preferred embodiment relates to the reprogramming of cell behavior through the manipulation of transcriptional regulators (TRs). The invention includes systemic treatment and compositions for such treatment, as well as in vitro manipulation of cells prior to transplantation of such cells with the host under treatment.
As will be discussed more fully hereinafter, a method is also disclosed for selecting target sites in the RNA transcripts of particular genes that individually have a high likelihood of being excellent target sites for the binding of antisense oligonucleotides which are intended to inhibit the expression of the corresponding gene. In a preferred embodiment, this target site selection method can be used to select antisense oligonucleotides for the treatment of Aberrant-Programming Diseases in accordance with the molecular model of Aberrant Programming Diseases disclosed herein. Taken together, this molecular model of Aberrant Programming Diseases (AP Model), which delimits the preferred gene targets for the development of the new therapies discussed herein for said diseases, and the method for selecting target sites within the transcripts of said genes, greatly simplifies the drug discovery process for the development of new treatment modalities and greatly increases the likelihood that clinically successful compounds will, thereby, be generated.
In yet another embodiment, the novel method disclosed herein for selecting antisense oligonucleotide target sites (and, thereby, the sequence of the corresponding antisense oligonucleotides) can be used with conventional rationale (independent of the AP model) by one with ordinary skill in the art to select therapeutic oligonucleotides for the treatment of a variety of diseases and medical purposes. The conventional rationale is, in essence, a rationale which links the known function(s) of particular molecules, in terms of their direct effects on specific cellular functions, with particular disease processes or therapeutic needs (as opposed to molecules such as TR that—non-obviously—may act indirectly as a result of being part of a combinatorial regulation mechanism).
2. Description of the Related Art
Very recent studies involving the use of antisense oligonucleotides for treatment of cancer have been reviewed by Stein and Cohen, Cancer Res. 48:2659 (1988). Several types of antisense molecules have been screened for their ability to inhibit the synthesis of particular proteins using both intact cells and in vitro systems for protein synthesis (See Ld. and Paoletti, Anti-Cancer Drug Design 2:325, 1988). For example, agents with specificity for RNA transcribed from the myc gene have been reported to inhibit the proliferation of the human AML line HL60 (Wickstrom, et al., Proc. Natl. Acad. Sci. USA 85:1028 (1988) and normal T lymphocytes (Heikkila, et al., Nature 328:445 (1987), and oligodeoxynucleotides complementary to cyclin mRNA have been reported to suppress the division of 3T3 cells (Jaskulski, et al. 1988).
More recently, it has been found that in the treatment of cancer with ODNs against myb, the proliferation of leukemic cells was inhibited with an accompanying lower degree of inhibition against normal cells. (Calabretta et al, PNAS, 88, 2351,1991.) Also, it has been shown that transient inhibition in a leukemia cell line resulted with an ODN against myc; however, unfortunately, a comparable inhibition against normal cells occurred (Zon et al patent). This patent also discloses inhibition of HIV replication using ODNs targeted to viral genes. Belenska et al (Science, 250, 997, 1990) have proposed the use of double stranded ODNs, binding to TR ligands as potential therapeutic agents for disease causing genes. They give blocking of NF-kB binding to HIV enhancer as an example. The use of retroviral vectors carrying antisense oncogenes for the treatment of cancer is known.
The fundamental problem with the foregoing part is that it is based on the notion that the expression of specific molecular abnormalities (altered regulation or mutation of endogenous genes or expression of exogenous genes) in the disease cells of these patients directly cause the clinical pathological features of the AP disease. It follows from such thinking that the therapeutic strategies should be directed to attacking these molecular abnormalities.
In the case of cancer, contemplated therapy involving antisense expression vector ODNs have been directed to oncogenes in accordance with the oncogene/anti-oncogene cancer model, or to growth factors expressed by cancer cells in accordance with the autocrine model. In the case of AIDS therapeutic strategies involving such agents being developed are directed toward blocking HIV expression and/or infection. There are no counterpart causal agents identified to the other AP diseases. Hence the therapeutic approaches under development are more empirical.
The present inventor first described in detail, in writing, in a confidential manuscript prepared for Gerald Zon, Ph.D. of Applied Biosystems Incorporated (now LYNX Therapeutics, Inc.) and completed in May of 1990, the concept of Aberrant Programming disease and the derivative notion of using antisense ODNs to modulate TR as a means of selectively reprogramming the cellular programs of the diseases cells/tissue in question. According to the AP disease model the fundamental pathology causing the clinical pathological features of these disorders is both relational and dynamic. In stark contrast to the prior art, the therapy of the present invention involves manipulation of patterns of TR expression. The invention provides an entirely new approach to the treatment of said selected diseases and provides a rational, empirical basis for the design of novel agents. The therapeutic reprogramming of normal tissue involving ODNs is unprecedented.
The Aberrant Programming model indicates that atherosclerosis is, for example, an Aberrant Programming Disease. In this case, atherosclerosis is said to result from a change in the pattern of expression of certain TR in the smooth muscle cells (SMC) associated with blood vessels; the changed pattern of expression of TR, then, is responsible for the particular differentiated state that characterizes atherosclerotic SMC and which, therefore, produces the disease. The conventional hypothesis that attempts to provide a molecular explanation for the pathogenic changes in atherosclerotic smooth muscle cells (SMC) is called the “monoclonal hypothesis” (Benditt and Benditt, Proc. Natl. Acad. Sci. USA 70: 1753, 1973). In essence, this hypothesis argues that atherosclerotic plaques are benign tumors that result from a mutagenic event in some key regulatory molecule, in a manner analogous to the conversion of a proto-oncogene to an oncogene in the case of malignant tumor cell development. In support of this “monoclonal hypothesis” it has been found that DNA isolated from cells recovered from atherosclerotic plaques is capable of transforming normal fibroblasts in a transfection-nude mouse assay, whereas DNA extracted from normal control endothelial cells does not induce such transformation. The gene(s) which are responsible for encoding this transforming capacity have not been identified, however. Ruled out so far are N-ras, K-ras, Ha-ras, erbA, erbB, fes, src, mos, Abl, sis, c-fos, c-myb and c-myc which have been shown to be expressed by SMC (Parkes et al., Am. J. Pathol. 138: 765, 1991). Thus, the “monoclonal hypothesis” requires the mutation of some key regulatory molecule as the causal factor in the development of atherosclerosis. It follows from the conventional rationale that this key regulatory molecule which is altered is the prime target for therapeutic intervention.
In contrast, the Aberrant Programming model argues that the basic molecular pathology in the atherosclerotic SMC is to be found in the pattern of TR expression, where the relevant TR are those involved in cellular program control. The Aberrant Programming model, therefore, identifies TR such as, for example, c-fos, c-myb and c-myc as being appropriate targets for evaluating potential therapeutic antisense ODNs for atherosclerosis in the Reprogramming Test (as defined hereinafter), even though these TR have not been found to be mutated in atherosclerotic SMC. If the molecular mutations that have been detected in the transfection-nude mouse assay contribute to the pathogenesis of atherosclerosis, they would be considered by the AP model to be “risk factors”. Risk factors in this context are defined as determinants that increase the probability that the afflicted cells/tissue will express an altered pattern of TR, thereby facilitating the generation of an Aberrant differentiation program. The AP model sets the foundation for a novel therapeutic strategy. The model predicts, for example, that there are antisense ODNs which, when targeted to certain TR, will produce a therapeutic reprogramming of atherosclerotic SMC (such as, for example, reversing the Aberrant cellular differentiation program to a more normal state, or, inducing apoptosis in the atherosclerotic SMC); the AP model also predicts that such effects will not be seen when the antisense ODN is used to treat a wide variety of other normal and diseased cell types that express different differentiation programs, even though they express the same TR target and the expression of said TR may be down regulated by the antisense ODN treatment. The basis of this logic can best be understood by making an analogy to “language”, as is done in Table I.
Rosenberg and his colleagues, using a rat model system, explored the potential use 30 of c-myb antisense ODNs for the treatment of restenosis. Restenosis refers to the re-occlusion of atherosclerotic blood vessels following a medical procedure to reverse the obstruction to blood flow produced by atherosclerotic plaques (Simons and Rosenberg, Circulation Res 70: 835, 1992; and, Simons et al., Nature 359: 67, 1992) (United States Patent Application #723454, 28 Jun. 1991; United States Patent Application #792146, 08 Nov. 1991; United States Patent Application #855416, 18 Mar. 1992). The second of the published studies demonstrates that the local delivery of phosphorothioate ODNs to rat carotid arterial SMC in vivo results in a substantial uptake of the ODNs by the SMC and a prolonged retention of these compounds by the SMC. These investigators showed that c-myb antisense ODN, but not the corresponding sense ODN, inhibited the proliferation of SMC in the aorta of normal animals following regional damage to the vessel wall resulting from balloon angioplasty. Balloon angioplasty damages the endothelium underlying the region of treatment and causes intimal migration and proliferation of the SMC over the length of the damaged blood vessel. The result of treating the damaged vessels with the c-myb antisense ODN was a substantial improvement in the patency of the affected vessel after the induced trauma, compared to control animals not treated with the c-myb antisense ODN. The c-myb antisense ODN used to treat the normal smooth muscle cells either in vitro or in vivo, however, had four guanine bases in a row which could cause the formation of a “G-quartet”, while the control ODN did not. The suppression of SMC growth, therefore, may not have been due to an antisense effect on c-myb, but rather from a non-antisense effect, with the reduction in c-myb expression in the SMC being a secondary event. This possibility was not apparently explored by these investigators.
Three groups have examined the possibility that c-myc antisense ODNs might be useful for the treatment of restenosis. Zalewski and his colleagues were the first to carry out these studies (Shi et al., Circulation 88: 1190, 1992; and Shi et al., Circulation 90: 944, 1994) (United States Patent Application #4799, published 7 Jan. 1993). The first of the Zalewski papers examined the usefulness of c-myc antisense ODNs for inhibiting the proliferation of normal human SMC. The c-myc antisense ODN was shown to inhibit the proliferation of the SMC while the corresponding sense ODN and a mismatched control ODN did not. The published in vivo work by Shi et al. (1994) again demonstrated that phosphorothioate ODNs can be readily delivered to SMC in the coronary vessels of animals where the ODNs are taken up in sufficient quantities to produce biological effects. Specifically, coronary blood vessels of pigs were damaged by balloon angioplasty. The human c-myc antisense ODN or the corresponding sense control ODN, were then applied to the damaged vessels. It was not reported, however, whether or not the human c-myc antisense ODN was sufficiently complementary to the c-myc transcript of the pig to permit effective binding of the human ODN sequence to the pig target transcripts. The c-myc, but not the control ODN, substantially inhibited the proliferation of SMC, resulting in improved blood flow through the affected vessels, compared to control animals not treated with the c-myc antisense ODN. Again, however, the antisense ODN used in the in vivo efficacy studies had four guanine bases in a row, while the control ODN did not. This four-guanine sequence could explain the capacity of the “therapeutic” ODN to inhibit the proliferation of the pig SMC and to inhibit the c-myc expression, while the “control” ODN did not. Bennett et al. (J. Clin. Invest. 93: 820, 1994) and Biro et al. (Proc. Natl. Acad. Sci. USA 90: 654, 1993), using the same c-myc antisense ODNs and control ODNS, demonstrated an inhibition of rat SMC proliferation in vitro.
Hence, the published studies of the use of c-myb or c-myc antisense ODNs to block experimentally-induced restenosis in animal models could be interpreted as showing that proliferation of cells (in this case, normal smooth muscle cells) can be blocked simply by exposure to compounds which have a non-specific capacity to inhibit proliferation (such as, for example, by non-specific masking of cell surface receptors, or by interference in essential metabolic pathways). Hence, these findings do not constitute “cellular reprogramming” for the purposes of achieving a therapeutic effect as defined herein. A “true reprogramming event” that involves inhibition of proliferation in accordance with the rationale provided herein would show a dependence on the differentiation status of the target cells; i.e., the reprogramming event (initiated by the antisense ODNs) must only work on cells that exhibit a particular set of differentiation programs, and not work on cells which exhibit a different set of differentiation programs. For example, an antisense ODN capable of blocking SMC proliferation by a reprogramming effect would not be able to block the proliferation of human cells in general. The possibility remains, however, that antisense ODNs directed to c-myc or c-myb could cause a therapeutic reprogramming of atherosclerotic SMC, in accordance with the present invention. This possibility remains because the appropriate experiments have not yet been done.
The design of antisense oligonucleotides for the inhibition of gene expression has been based primarily on one or the other (or both) of two considerations. First, investigators have targeted antisense oligonucleotides to regions of RNA transcripts known to be involved in the control of pre-mRNA processing or mRNA translation, such as splice sites or the start codon (AUG), respectively. Second, investigators have used computer models of the secondary structure of mRNA to “visualize” mRNA regions that might be susceptible to ODN targeting; these structural modeling procedures are not, however, highly predictive of the actual secondary structure of the mRNA in situ. Design of antisense ODNs according to novel methods disclosed in the present invention, however, is not dependent on either of these approaches to antisense ODN design. Rather, disclosed. herein is a novel computer-based method for selecting unique “hotspots” in RNA transcripts that are particularly well suited for targeting antisense oligonucleotides for the purpose of inhibiting the expression of genes and thereby greatly enhancing the likelihood of producing therapeutic effects. The method herein described appears to be an unexpected and substantial improvement over the two conventional approaches to selecting target sites for antisense oligonucleotides.
There are now many examples of the successful use of antisense ODNs to selectively block the expression of any of a wide variety of gene targets, both in in vivo and in in vitro studies. For example, in in vivo model systems: inhibition of Human Immunodeficiency Virus (HIV) gene expression (including tax gene) in human cells grown as xenogeneic transplants in animal models (Kitajima et al., J. Biol. Chem. 267: 25881, 1992);targeting genes in xenotransplanted human cancer cells in animal models, including targeting c-myc, c-Ha-ras, NF-KB, c-myb, c-kit and bcr-Abl (Agrawal et al., Proc. Natl. Acad. Sci. USA 86: 7790, 1989; Agrawal et al., Proc. Natl. Acad. Sci. USA 88: 7595, 1991; Biro et al., Proc. Natl. Acad. Sci. USA 90: 654, 1993; Gray et al., Cancer Res. 53: 577, 1993; Higgins et al., Proc. Natl. Acad. Sci. 90: 9901, 1993; Ratajczak et al., Proc. Natl. Acad. Sci. USA 89: 11823, 1992; Wickstrom et al., Cancer Res. 52: 6741, 1992; Skorski et al., Proc. Natl. Acad. Sci. USA 91: 4504, 1994). In each of these instances involving the administration of antisense ODNs to treat animals with xenogeneic human cancers, the transplanted malignant cells were found to regress.
A number of difficulties, however, have also been reported in the use of antisense ODNs in in vitro studies, none of which have proven to be insurmountable, however, in view of the existing art and technology. In general, problems in the in vitro use of antisense ODNs (most commonly phosphorothioates) have centered around what has been viewed as “poor uptake” and/or the production of unintended biologic effects; i.e., non-antisense effects. These unintended effects fall into two major categories: first, there are biologic effects that are attributable to the backbone structure of the oligonucleotides; and, second, there are sequence-specific non-antisense (aptameric) effects that appear to be dependent upon the three-dimensional conformation of the ODN in solution, and, consequently, on the positioning of the molecular electrostatic (ionic) charges associated with the ODN molecule. Like ODN antisense effects, both of these non-antisense effects are dose dependent.
There are large differences in the capacity of similar ODNs directed to transcripts of a given gene to block the expression of that gene in cells; the reasons appear to be related to variations in the availability of the particular target site on the transcript complementary to the antisense ODN. No method has previously been described which permits antisense ODNs to be designed so that, with a high probability, some will exhibit optimal activity in the purpose intended. Hence, what has been referred to as the “poor uptake” of ODNs by some cell types in vitro may in large part reflect the use of antisense ODNs that are not properly designed and are, therefore, not optimally potent. It is also possible that the culturing of cell lines under atmospheric oxygen conditions (which is the usual and common in vitro practice) produces a situation in which antisense ODNs are made less active than they may be at much reduced (and more physiologically-relevant) oxygen tensions (Smith L J and Kay H D. Unpublished observations). The basis of this latter phenomenon could be due, at least in part, to the increased generation of reactive free oxygen radicals under ambient (atmospheric) oxygen levels by cells following treatment with any of several types of ODNs, such as phosphorothioates. Highly-reactive free oxygen radicals have been shown to have the capacity to alter the lipids in the surface membranes of cells, and to activate certain second-messenger pathways. Such alterations could lead to an inhibition of antisense ODN uptake and/or to other non-antisense ODN-dependent biologic effects.
A complete blockade of the induction of free radical formation by cells in response to exposure to ODNs at physiologic oxygen levels would require the presence of potent anti-oxidants such as, for example, vitamin C or vitamin E. Finally, in general, it appears that antisense ODNs are more active when used on freshly-obtained patient specimens than they are when used on established cell lines, either in vitro or in vivo. Furthermore, at least some established cell lines appear to be more responsive to antisense ODNs when studied in vivo in animal models than when studied in vitro in cell cultures. Dean and McKay (Proc Natl Acad Sci USA 91: 11762, 1994), for example, found that an antisense ODN directed to PKCa could inhibit the growth of the C127 murine mammary epithelial cell line both in vitro and in vivo. To get enough ODN into the cells grown in vitro to reduce PKCa expression and inhibit growth, cationic liposomes had to be utilized. The naked antisense ODN, however, worked very well when injected into mice carrying the C127 cell line as a transplant. Again, this greatly-superior in vivo response is consistent with the concept that the ODNs cause a much lower level of free radical production in the animal.
Similarly, the apparent unintended backbone-dependent biologic effects of antisense phosphorothioate ODNs on treated cells can be eliminated (or adequately reduced) by the use of more appropriately designed (and, therefore, more efficacious) antisense ODNs. These unintended biologic effects, of which the inhibition of cell proliferation is most common, generally only occur at phosphorothioate concentrations of 5-10 micromolar (μM) or greater in the final culture medium. The better designed and more potent antisense ODNs, however, are biologically most active at least 10-fold lower concentrations, particularly when fresh tissue is used, or when the antisense ODN is used in vivo.
Pronounced aptameric effects usually appear to be the property of only a small proportion of ODN. Aptameric effects result when an ODN binds tightly and specifically to a particular biomolecule, and, as a result, modifies the biological function/behavior of said biomolecule. Aptameric effects are dependent on the nucleotide sequence in the ODN. Presumably, the sequence dependence of these effects reflects the fact that ODNs with different nucleotide sequences assume different spacial conformations, dictated by neighboring nucleotide-nucleotide interactions. The nature of the backbone chemistry, however, is also relevant in aptameric effects since said chemistry (and associated molecular electrostatic [ionic] charges) also influences the overall spacial conformation which the ODN molecule can assume in solution. Only a subset of the possible aptameric effects which an ODN might produce, however, would be expected to be an absolute counter-indication for therapeutic use as an antisense compound. Any such undesirable effects can be overcome by simply choosing another antisense ODN directed to the same target transcript, but which contains a different nucleotide base sequence, or, in some instances, by changing the ODN backbone. The former option may involve selecting an entirely different “hotspot” on the transcript, or simply making modest changes (length, position) in the ODN in question. Changes in ODN secondary structure may also be achieved by making a small number of base substitutions, such as with inosine, that do not interfere significantly with binding of the ODN to the target RNA transcript.
In contrast, some antisense ODNs may possess aptameric-like effects that enhance their therapeutic efficacy. The present inventor, for example, has found that some antisense ODNs (in particular, phosphorothioate ODNs) which target MDR1 gene transcripts (and thereby inhibit P-glycoprotein expression) apparently can also reduce MDR1 mRNA levels by an aptameric-like effect that presumably involves the inhibition of second messenger pathways such as the protein kinase-C and/or protein kinase A pathways.
Many of the in vitro successes in the application of antisense ODNs for therapeutic purposes have been readily extrapolated to in vivo use. This is evidenced by the numerous publications showing the in vivo efficacy of antisense ODNs. Furthermore, several ODNs have already been approved by the United States Food and Drug Administration for clinical testing. It should be noted that the ODN uptake problems sometimes encountered in in vitro studies have not been reported to be problematic in in vivo studies. Pharmacologic/toxicologic studies of phosphorothioate antisense ODNs have shown that phosphorothioates are adequately stable under in vivo conditions, and that they are readily taken up by all the tissues in the body following systemic administration (Iversen P I, Anticancer Drug Design 6:531, 1991; Antisense Res Develop 4:43, 1994; Crooke, Ann Rev Pharm Toxicol 32: 329,1992; Cornish et al., Pharmacol Comm 3: 239,1993; Agrawal et al., Proc Natl Acad Sci USA 88: 7595, 1991; Cossum et al., J. Pharm. Exp. Therapeutics 269: 89, 1994). In addition, these compounds readily gain access to the tissue in the central nervous system following injection into the cerebral spinal fluid (Osen-Sand et al., Nature 364: 445,1993; Suzuki et al., Amer J. Physiol. 266: R1418,1994; Draguno et al., Neuroreport 5: 305, 1993; Sommer et al., Neuroreport 5: 277, 1993; Heilig et al., Eur. J. Pharm. 236: 339, 1993; Chiasson et al., Eur J. Pharm. 227: 451, 1992). Phosphorothioates per se have been found to be relatively non-toxic, although a few particular ODNs have produced unintended toxic effects in animals. The latter instances of toxicity seem to be attributable to an unexpected aptameric effect on the part of the ODN in question.
In summary, it appears that antisense ODNs have the essential properties which make them useful as therapeutic agents, both in vivo and in vitro. In vitro antisense activities now can reasonably be expected to be seen in vivo. Two major areas needed for further development of antisense ODNs as therapeutic agents involve (a) the choice of gene targets for diseases like cancer and atherosclerosis, Alzheimer's and schizophrenia, and (b) methods for the selection of optimally active antisense ODNs directed to a particular gene target. These needs are addressed by the novel inventions herein described in the present invention.